Project Summary Epigenetic modifications, such as DNA methylation and histone modification, alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. In animals and plants, the strategies used to accurately target, maintain, and modify patterns of DNA methylation are mechanistically similar. The long-term goal of my study is to discover and characterize RNAs and proteins involved in the establishment of epigenetic modifications. My short-term interest is to investigate the RNA-directed DNA methylation (RdDM) pathway in plants. The RdDM pathway requires two plant-specific RNA polymerases, Pol IV and Pol V. Pol IV acts in partnership with a RNA-dependent RNA polymerase, RDR2, to transcribe DNA into short double-stranded RNAs (dsRNAs). These dsRNAs are then diced by the DICER endonuclease, DCL3, yielding 24 nt short interfering RNAs (siRNAs). The siRNAs are loaded into an Argonaute protein, primarily AGO4 or AGO6. AGO4-siRNA complexes are recruited to sites of Pol V transcription, where siRNAs are thought to basepair with Pol V transcripts, and AGO4 can bind to the C-terminal domain of the Pol V largest subunit. The DNA methyltransferase, DRM2 (the ortholog of human DNMT3), is subsequently recruited to methylate cytosines within the Pol V-transcribed DNA, accompanied by histone modifications that cause gene silencing. A detailed mechanistic understanding of the RdDM process remains elusive due to a lack of biochemical evidence to support what is mostly genetic evidence. By understanding whether the proposed alternative interaction modes of AGO4-siRNA, with Pol V transcripts, or Pol V itself, are mutually exclusive or not, my studies will provide new information about how noncoding RNAs and silencing complexes are targeted to chromatin sites. My specific aims are to develop methods for loading siRNA into AGO4 in vitro and understand the relative contributions of RNA-RNA and protein-protein interactions in AGO4- siRNA associations with Pol V transcription complexes, using biochemical assays and EM structural analyses. Because DNA methylation serves as a stable epigenetic mark to prevent gene activation during development, and alteration of DNA methylation and chromatin modification is implicated in human diseases, including cancer, an understanding of how noncoding RNAs and enzymes of the RdDM pathway coordinate gene silencing will yield new insights into fundamental processes important for human development and disease.